Methylation of the RIZ1 Gene In Myelodysplastic Syndrome and Acute Myeloid Leukemia

Abstract Abstract 5112 Inactivation of a tumor suppressor gene is often caused by a mutation, small deletion of one allele accompanied by loss of the second allele. Methylation in a promoter CpG of several tumor suppressor genes has recently been reported and has been associated with loss or decreased expression in many tumors. We previously reported frequent loss of heterozygosity on the short arm of chromosome 1 (1p) in the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML). The retinoblastoma protein-interacting zinc finger gene RIZ maps to 1p36. Mouse gene knockout models show that RIZ1 inactivation can cause tumor susceptibility. Inactivation of the RIZ1 gene by promoter hypermethylation has been reported in breast, liver, and gastric carcinoma. Previous study showed altered expression of the RIZ1 gene in human leukemia. However, methylation status of the RIZ1 gene has not been well studied in hematological neoplasms. To determine the relevance of the RIZ1 methylation, we performed methylation specific-polymerase chain reaction (PCR) analysis on the RIZ1 gene in 34 patients with MDS and 17 with AML evolved from MDS (secondary AML) as well as 55 patients with de novo AML. The 34 MDS samples consisted of 13 refractory anemia (RA), 1 RA with ringed sideroblasts (RARS), 10 RA with excess of blasts (RAEB), 6 RAEB in transformation (RAEB-t), and 4 chronic myelomonocytic leukemia. The 55 de novo AML consisted of 1 M0, 12 M1, 17 M2, 7 M3, 8 M4, 7 M5, 1 M6, and 2 M7. Written informed consent was obtained from the patients. Methylation of the RIZ1 gene was detected in 17 of the 34 MDS (50%) and 22 of 72 de novo and secondary AML (31%) (p=0.053). Methylation was detected in 7 of 14 low risk MDS (50%) and 10 of 20 high risk MDS (50%). Patients with MDS were classified using the IPSS score. Frequency of methylation was not statistically different among IPSS subgroups (p=0.419). No statistical differences were observed between methylation and overall survival (3 years) or progression to AML. In AML patients, methylation was more frequent in secondary AML (11/17, 65%) than in de novo AML (11/55, 20%) (p=0.0005). To define the methylation status of the CpG in the RIZ1 promoter region, we performed bisulfite sequence in several samples with methylation. Bisulfite sequence analysis revealed methylation at many CpG sites in the promoter region. Expression of the RIZ1 gene was examined by quantitative real time reverse transcriptase-PCR analysis in 22 samples of MDS and AML. RIZ1 expression (mean) was not statistically different in secondary AML and de novo AML (2.026 vs. 1.900, p=0.815). RIZ1 expression (mean) was not statistically different in methylation-positive group and methylation-negative group (1.996 vs. 1.810, p=0.728). In comparison with expression of normal bone marrow cells, decreased RIZ1 expression was accompanied by methylation in 6 of 9 samples examined, while it was also observed in 7 of 13 without methylation. HL-60 myeloid leukemia cells with RIZ1 methylation were cultivated for 3 days in the presence of various concentrations of 5-Aza-dC. Treatment of the leukemia cells with 5 Aza-dC induced growth suppression with RIZ1 restoration. Our results suggest that the RIZ1 gene was inactivated in MDS and AML in part by methylation, whereas another mechanism of inactivation should be involved in others. Disclosures: No relevant conflicts of interest to declare.